Age-related macular degeneration (AMD) is a common cause of visual loss in the aging human population. Drusen production by retinal pigment epithelium (RPE) is an important hallmark of the disease, as it precedes the appearance of visual symptoms. Studies have shown the presence of Drusen in both normal and diseased aging humans. However, what molecular events underlie Drusen formation and ultimately trigger the onset of disease are not understood. Recently, TIMP-3 (tissue inhibitor of metalloproteinase) has been identified as a candidate gene in a related retinal disease, Sorsby's Fundus Dystrophy. In preliminary experiments, we have observed increased expression of MMP-2 protein in eyes of AMD-affected patients, relative to normal human donors. In the proposed study we will test the hypothesis that alterations in the levels and/or activity of TIMP-3 and/or MMP-2 alter the production of extracellular matrix by RPE. First, we will screen RPE from normal and AMD-affected human donors and a cell line for the expression of known MMP and TIMP mRNA and proteins, using immunohistochemistry, in situ hybridization, PCR and zymography. Next, we will individually express TIMP-3 and MMP-2 cDNAs in a human RPE cell line and monitor effects of over expression on RPE and matrix production by these cells. We will also test if over expression produces alterations in a pre-existing ECM to result in Drusen-like deposits. The effect of TIMP-3 to MMP-2 ratio will also be examined by over expression of MMP-2 in a TIMP-3 expressing RPE cell line. These approaches will be extended to other MMPs and TIMPs identified as important in the first step.